DNA interstrand crosslinking plays a critical role in the action of certain anti-tumor agents and environmental toxins and also in the pathobiology of endogenous DNA damage as represented by Fanconi anemia (FA). DNA crosslinking cancer chemotherapeutics, including mitomycin C, nitrogen mustards, and platinum compounds, utilize the greater sensitivity of actively growing cancer cells to crosslinking agents. FA, a rare inheritd genetic disease, is caused by mutations in genes regulating replication-associated removal of interstrand DNA crosslinks (ICLs). By linking two DNA strands, ICLs inhibit both DNA replication and transcription. Failure to remove ICL lesions ultimately leads to cell death. Moreover, hereditary breast and ovarian cancers are triggered in individuals with genetic defects in the BRCA genes of FA/BRCA ICL repair pathway. Therefore, it is very critical to reveal the ICL repair mechanism to improve cancer chemotherapy and understand human genetic disease and hereditary cancer proneness. It has long been believed that progression of a replication fork is invariably inhibited when a replication complex encounters an ICL and that fork progression resumes only after the ICL is repaired. However, our recent experiments on ICL repair taking place at a replication fork produced an unexpected result, which is inconsistent with this widely endorsed hypothesis. Our results suggest the formation of a new DNA replication fork downstream of ICL and, additionally, indicate that the recently discovered human Primase-Polymerase, PRIMPOL, plays a critical role in this event. Confirmation of this revolutionary new hypothesis and the demonstration of PRIMPOL's essential role in this process are expected to open important new avenues in our understanding of the mechanism of mammalian ICL repair. To achieve this goal, a single ICL is inserted into plasmid that replicates in one direction in human cells synchronously with the replication of host cells. Progeny plasmids are recovered from cells and analyzed for repair events, which serve as a marker for a new fork formation. The role for PRIMPOL in the formation of a new fork is evaluated by knocking down its activity by the RNA interference technology. For detailed studies, the PRIMPOL gene is destroyed by a newly developed gene knockout technology, and knockout cells are complemented with PRIMPOL mutants. Achievements of these aims will contribute to the improvement of cancer chemotherapeutic drugs and the full understanding of the human genetic diseases associated with a defect in ICL repair.